高静水压刺激对血小板活化的影响及PPARγ的保护作用的研究
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摘要
研究背景和总体思路
已经明确高血压病患者血小板聚集性和粘附性均明显亢进。由于相关研究多集中于临床观察,但高血压病患者常合并多种异常情况,除血压外,其他并存的因素也参与血小板激活。因此血压本身对血小板激活的作用及其程度尚不明确。为此,我们在离体孵育的条件下,观察静水压对血小板的影响。
最近研究发现,血小板不仅能合成蛋白质,还通过剪切预存于多聚核糖体中的未成熟的RNA为成熟的mRNA,翻译mRNA合成蛋白质。当血小板激活时起动了细胞内的信号转导反应,导致特异性的RNA被翻译并产生蛋白质。由于血小板源于巨核细胞,巨核细胞的mRNA也能直接影响血小板激活后的功能。我们设定,高血压病患者的血小板的异常变化,部分可能是巨核细胞变化的继发变化。目前,高血压对外周循环巨核细胞是否影响,是否会进而影响血小板的状态及功能,尚未见研究报道。
血小板含有高水平的过氧化物酶体增殖物激活受体γ(Peroxisome Proliferator Activated Receptor, PPARγ)。PPAR的作用广泛,涉及多方面。研究证实血小板的PPARγ具有活性,能抑制血小板的激活。对于高血压病患者血小板PPARγ的变化,尤其是PPARγ的信号转导途径,值得深入研究。
基于上述未决问题,本课题利用自制的高静水压装置,在体外利用不同的压力条件孵育血小板及巨核细胞,进行以下几方面的研究:高静水压对血小板的形态和结构及功能的影响、高静水压对巨核细胞的形态和结构及功能的影响和PPARγ在高静水压刺激血小板中信号传导作用。通过上述研究,以加深高血压对血小板激活认识,为临床更有效地防治高血压靶器官损害提供有价值的实验依据。
第一部份高静水压对血小板的形态和结构及功能的影响
目的:
利用体外静水压孵育血小板的模型,观察不同压力,以及同一压力不同时间对血小板的形态、结构及功能的影响。
方法:
建立高压培养的模型后,以180mmHg处理血小板不同时段(0h、1h、6h和18h)和不同静水压(0mmHg、120mmHg、180mmHg和240mmHg)处理血小板18小时,观察以下指标。1、在电镜下观察血小板超微结构的变化,全自动血细胞分析仪测定血小板形态参数,全自动生化分析仪测定血小板孵育液中LDH;2、测定血小板聚集率和流式细胞仪测定血小板活化表面标志物PAC-1和CD40L(CD154);3、免疫印迹法测定血小板内CD40L(CD154)、P-selectin、PPARγ和Caspase 3的蛋白表达;4、血小板PPARγ活性的测定。
结果:
1、血小板超微结构:正常大气压孵育血小板18小时,血小板细胞呈静止状态,细胞规整、表面突触少、致密颗粒多;高静水压刺激后,细胞形态大小不一,血小板羽状突起增多。细胞内致密颗粒减少,血小板呈活化状态,个别细胞出现消解、崩溃的图形。
2、血小板的参数:正常大气压孵育血小板不同时段的血小板的参数无明显差异。180mmHg静水压处理下6小时和18小时,平均血小板体积(MPV)显著减少,18小时血小板分布宽度(PDW)的显著增加。不同的静水压处理血小板,240mmHg静水压组血小板数(PLT)的显著降低;与对照相比,180mmHg和240mmHg引起血小板MPV、PDW的显著减少。
3、LDH的变化:正常大气压孵育各时段的上清中的LDH无统计学差异,而180mmHg处理组,随时间延长,LDH浓度逐渐增加,18小时段明显高于其他时段。不同静水压处理组,240mmHg、180mmHg处理组均明显高于对照组。
4、血小板聚集率:180mmHg刺激1小时后最大聚集率即明显增加,6小时和18小时的2min、4min和最大聚集率均有极显著的增加。不同压力组的比较可见,180mmHg和240mmHg处理组的2min、4min和最大聚集率均比对照组显著增加,180mmHg组的2min、4min和最大聚集率均显著高于120mmHg组,但240mmHg组的聚集率较180mmHg组有所下降,但无统计学意义。
5、血小板PAC-1:三个高静水压处理组血小板的PAC-1均显著增加;在180mmHg处理1小时血小板激活有增加但不显著,在6小时和18小时相较0小时和1小时均可检测到显著的血小板激活。
6、P-selectin蛋白表达:不同静水压的比较表明,随着静水压的增加,P-selectin表达逐渐增加。180mmHg和240mmHg的P-selectin的表达显著高于120mmHg组。
7、CD154的表达:血小板胞浆裂解液中CD40L(CD154)的量在180mmHg和240mmHg有显著性降低;流式细胞仪检测血小板膜表面CD40L(CD154)的表达随着静水压的增加而明显增加。
8、血小板内PPARγ的蛋白量:不同压力组之间,同一压力不同时间组之间的血小板内PPARγ的蛋白量无统计学差异。但PPARγ活性随着压力的增加(120mmHg和180mmHg)而显著增加,但240mmHg组PPARγ活性与180mmHg则明显的降低。
9、血小板内caspase 3的蛋白表达:随着压力的增加,血小板内caspase 3的17KD片段蛋白表达逐渐出现并增高,同时32KD的量显著性减少。180mmHg和240mmHg17KD的量比对照和120mmHg有显著增加。
结论:
1、随着压力增高,血小板形态发生明显改变,表现为平均血小板体积(MPV)显著减少,分布宽度的增加;在180mmHg静水压下,随着时间的延长也出现类似改变。
2、较高的高静水压(180mmHg和240mmHg)使活化的GPIIb/GPIIIa复合体(PAC-1)增多,促使血小板活化和聚集。
3、高静水压(180mmHg和240mmHg)可使血小板内P-slectin的表达量增加,其胞浆的CD40L(CD154)裂解,胞膜上CD40L(CD154)增加,血小板中炎症相关分子释放增加,促进炎症反应。
4、静水压刺激的血小板中PPARγ蛋白含量无明显变化,但其活性随着压力和时间的增加而有一定幅度的增加。
5、180mmHg和240mmHg刺激血小板18小时导致血小板破坏,机制与高静水压激活血小板Caspase 3,导致血小板凋亡有关。
第二部份高静水压对巨核细胞的影响
目的:
研究静水压对巨核细胞系Meg-01形态、结构及血小板功能相关基因表达的影响,探讨高静水压对巨核细胞功能的影响。
方法:
建立巨核细胞高压培养的模型,分别用不同的压力(0、120、180、240mmHg)刺激Meg-01细胞,用MTT法检测细胞的增殖率,TUNEL法检测细胞凋亡百分比,RTPCR测定GPIIb、GPIIIa、P-selectin和PPARγ基因表达。
结果:
1、增殖率:120mmHg组与正常对照组相比,细胞有明显增殖,180mmHg组和240mmHg组与对照相、120mmHg组比,细胞增殖率有明显下降,且180mmHg组和240mmHg组二组间的细胞增殖率也有显著性降低。
2、凋亡率:20mmHg处理组的凋亡指数与正常对照组无显著性差异,而静水压达180mmHg和240mmHg时细胞的凋亡率显著增加。
3、GPIIb/GPIIIa的mRNA的量高静水压处理巨核细胞系Meg-01细胞后,细胞内GPIIb的mRNA的量180mmHg和240mmHg处理组与正常对照组和120mmHg有明显增加。细胞内GPIIIa的mRNA的量180mmHg和240mmHg处理组与正常对照组和120mmHg有明显增加,240mmHg较180mmHg也有显著增加。
4、P-electin的mRNA量:P-electin的mRNA量在静水压180mmHg显著性增加,240mmHg虽有增加,但无显著性意义。
5、PPARγ的mRNA量:细胞在120mmHg刺激下,PPARγ的mRNA量有显著性增加,而在180mmHg和240mmHg的压力下PPARγ的mRNA量相较对照组无显著性变化。
结论:
1、120mmHg静水压可促进Meg-01细胞增殖,而180mmHg和240mmHg则抑制巨核细胞增殖,促进其凋亡。
2、180mmHg和240mmHg静水压刺激促进Meg-01细胞表达GPIIb/GPIIIa和P-slectin。
3、120mmHg静水压下PPARγ表达增强,在更高静水压则无变化。
第三部份PPARγ对高静水压处理血小板的保护作用
目的:
探讨PPARγ在高静水压处理的血小板中保护作用。
方法:
实验分为C、C+wy、C+gw、H、H+wy和H+gw六组(C为正常大气压孵育,H为180mmHg静水压孵育,wy为PPARγ激动剂,gw为PPARγ阻断剂),处理18小时后检测各组血小板聚集率,血小板内钙离子浓度,血小板内和孵育基中NO的量,血小板内血小板内的iNOS、eNOS、CD40L(CD154)、NFκB和P-selectin蛋白量的表达。
结果:
1、血小板聚集率:C、C+wy、C+gw三组的血小板聚集率无统计差别。H组2min、4min和Max聚集率均显著高于C组; H+wy组的三种聚集率显著低于H组;H+gw组比H组有升高,但无统计意义。2、PAC-1:C、C+wy、C+gw三组的血小板膜PAC-1无统计差别。H组的PAC-1比C组显著升高;H+wy组的PAC-1比H组显著降低;H+gw组的PAC-1较H组高,但无统计意义。
3、CD40L(CD154)的表达:C、C+wy、C+gw三组的血小板膜CD40L(CD154)和细胞内CD40L(CD154)的表达无统计差别。H组血小板膜上CD40L(CD154)的表达比C组显著升高,细胞内的CD40L(CD154)比C组明显降低;H+wy组血小板膜上CD40L(CD154)比H组显著降低,细胞内的CD40L(CD154)比C组明显升高;H+gw组血小板膜上的CD40L(CD154比H组高,细胞内的CD40L(CD154)比H组低,但均无统计意义。
4、血小板内钙离子浓度:C、C+wy、C+gw三组的血小板内钙离子浓度无统计差别。H组血小板内钙明显高于对照组;H+wy组的钙离子明显低于H组,但仍显著高于对照组;H+gw组的钙离子明显高于对照组,比H+wy组高,但无统计意义。
5、血小板的上清中和血小板内的NO:C、C+wy、C+gw三组的血小板的上清中和血小板内的NO无统计差别。H组血小板的上清中和血小板内的NO显著低于对照组;H+wy组和高静水压组相比NO均有显著性升高,和正常对照组相比上清和细胞内的NO仍有明显降低;H+gw组明显低于对照组和H+wy组,和H组无显著性差别。
6、血小板内iNOS和eNOS的表达:C、C+wy、C+gw三组的血小板内iNOS和eNOS的表达无统计差别。H组的iNOS明显高对照组,eNOS显著下降;H+wy组与H组相比,eNOS明显升高,iNOS显著下降;H+gw组eNOS较H组低,但无统计意义,iNOS明显高于H组。
7、血小板P-selectin、表达:C、C+wy、C+gw三组的血小板P-selectin的表达无统计差别。H组的P-selectin明显高于C组;H+wy组、H+gw组与H组相比, P-selectin表达无显著性差异。
8、血小板内NF-κB的表达:C、C+wy、C+gw三组的血小板NF-κB的表达无统计差别。H组的NF-κB明显高于以C组;H+wy组、H+gw组与H组相比, P-selectin表达无显著性差异。
结论:
1、在180mmHg刺激18小时的血小板,PPARγ的激动剂Wy 14643能显著降低血小板聚集和活化,降低血小板膜上PAC-1的表达,可能通过降低血小板内钙离子浓度,升高血小板内的eNOS的表达,降低iNOS的表达,而升高血小板内和孵育液中的NO的量这两条途径。
2、在180mmHg刺激18小时的血小板,PPARγ的阻断剂Gw 9662并不能进一步激活血小板,可能血小板中PPARγ已经处于高度抑制状态有关。
3、PPARγ的激动剂Wy 14643显著降低血小板膜上CD40L(CD154)的表达,稳定胞浆中的CD40L(CD154);而对P-selectin没有明显的改变,提示血小板内的PPARγ调节血小板的免疫功能可能主要通过调节CD40L的量来调节,而对P-slectin和NF-κB作用不显著。
Background and design
Findings have already revealed that both platelet clumping and cohesion are sthenic (overactive) in patients with hypertension. Pertinent researches on the relationship of hypertension and platelet mainly focus on clinical observation. However, other factors besides blood pressure can also be involved in platelet activation as hypertension patients often suffer from many other abnormal conditions. Thus, the effect of hydrostatic pressure on platelet was tested in vitro in order to determine platelet activation and its extent influenced by the single factor of blood pressure.
Recent researches found that platelets can not only synthesize proteins but also translate mRNA into proteins via splicing pre-mature mRNA pre-stored in polyribosome into mature mRNA. When Platelet activated, signal transduction reaction starts up and results in subsequent specific RNA translating and protein synthesis. As platelet originates from megakaryocyte, megakaryocyte mRNA has a direct influence on the functions of activated platelet. The changes of platelet in hypertension patients could be a subsequent event of megakaryocytes alternation. However, it has not been studied yet, whether high pressure can influence peripheral vascular megakaryocytes and further affect platelet states and functions.
Peroxisome proliferator activated receptors (PPARs), which have a wide range of diverse functions, expresses highly in Platelet. Studies showed that platelet PPARγis active and can prohibit the activation of platelet. Platelet PPARγchanges in hypertension patients and the signal transduction ways via which PPARγcan exert its influence deserve further study.
Based on the unresolved problems mentioned above, the present research firstly cultured platelet and megakaryocyte in vitro under different hydrostatic pressure(a self-designed high-hydrostatic-pressure device is made to treat platelet under different hydrostatic pressure) and then studied in the following respects: high hydrostatic pressure’s influence on the shape, structure and function of both platelet and megakaryocyte; the protection of PPARγafter platelet has been intrigued by high hydrostatic pressure. This study aims to enhance the understanding of activation of platelet under high blood pressure so as to evaluate therapeutic effect of antihypertensive in new perspective. The experimental data could provide valuable basis for more effectively clinical prevention and treatment of damage in target hypertensive organ
Section one: influence of high hydrostatic pressure on platelet shape, structure and function
Objective:
By utilizing the model of culturing platelet under high hydrostatic pressure in vitro and treating platelet under 180mmHg hydrostatic pressure for 4 different period of time(0h,1h,6h and 18h) or treating platelet for 18h but under 4 different hydrostatic pressure(0mmHg,120mmHg,180mmHg and 240mmHg), changes of platelet shape, structure and function have been studied.
Method:
Firstly the model of platelet culturing under high hydrostatic pressure has been established. Then platelet has been treated under 180mmHg hydrostatic pressure for 4 different period of time(0h, 1h, 6h and 18h) or treated for 18h but under 4 different hydrostatic pressure (0mmHg, 120mmHg, 180mmHg and 240mmHg), and the following indices has been tested: 1) platelet ultrastructure changes were observed by electron microscope, platelet shape parameters were determined by full automatic blood cell analyzer, LDH in platelet culture medium detected by full automatic biochemical analyzer; 2) platelet aggregation rate, 3) PAC-1 and CD40L(CD154) (surface marker of platelet activation) determined by flow cytometry; 4) the expression of CD40L(CD154), P-selectin, PPARγand Caspase3 in platelet determined by immunoblot; 5) PPARγactivity.
Results:
1) Platelet ultrastructure: after 18h culturing under normal atmosphere pressure, platelet cells are of the similar shape and size and in a static state with a small number of dendritic extensions and many dense granule while after high hydrostatic pressure treatment, platelet cells become different in shape and size with an increasing number of dendritic extensions. With the decrease of dense granule platelet becomes active and some specific platelet cells take on a picture of breakdown.
2) Platelet parameters: have no significant difference among various time-treatment groups (all under the same atmosphere pressure). When treated for 6h or 18h under180mmHg hydrostatic pressure the mean platelet volume(MPV) declined dramatically , the platelet component distribution width(PDW) rose remarkably in the 18h time-treatment group; among the different hydrostatic pressure treatment groups, PLT dropped essentially in 240mmHg treatment group; compared with the control group, MPV and PDW decreased dramatically when treated in 180 mmHg and 240mmHg.
3) LDH changes: when platelet cultured under normal atmosphere pressure LDH in upper clear solution among different time-treatment groups have no statistic difference. However, when cultured under180mmHg hydrostatic pressure, LDH concentration increases gradually and its value in 18h time-treatment group is remarkably higher than other groups. Among different hydrostatic pressure treatment groups, LDH concentration in both 240mmHg and 180mmHg treatment groups is notably higher than control group.
4) Platelet aggregation rate: after 1 hour treatment of 180mmHg hydrostatic pressure the largest aggregation rate enhances greatly and after 6 hour or 18 hour treatment the 2min, 4min and largest aggregation rate elevate significantly. Among different hydrostatic pressure groups (180mmHg and 240mmHg), the 2min, 4min and largest aggregation rate increased significantly and all three aggregation rates of 180mmHg group are significant higher than those of 120mmHg group. However, the maximal aggregation rate of 240mmHg group is smaller than that of 180mmHg group but with no statistical significance.
5) Platelet PAC-1: PAC-1concentration in all three hydrostatic pressure groups elevate greatly; after 1 hour treatment of 180mmHg hydrostatic pressure, the activation of platelets increase but not significant while after 6 hour or 18 hour treatment remarkable platelet activation can be detected when compared with 0 hour or 1 hour treatment.
6) As the elevation of hydrostatic pressure, the volume of both P-selectin protein increases gradually: P-selectin expression level in 180mmHg and 240mmHg group increases more dramatically than that of 120mmHg group.
7) CD154 expression: The quantity of CD40L (CD154) in platelet lyses in 180mmHg and 240mmHg group declines notably; Tests by flow cytometry show that CD40L (CD154) expression in platelet surface increases remarkably with hydrostatic pressure elevating.
8) PPARγexpression in platelet: PPARγexpression levels among different pressure-treat groups and different time-treat groups show no statistical difference. Yet, PPARγactivity augmented remarkably when pressure rose from 120mmHg to 180 mmHg. However, PPARγactivity under 240mmHg is greatly lower than that of 180mmHg group.
9) Expression level of caspase 3 in platelet: with pressure elevation 17KD fragment of caspase 3 gradually emerges and accumulates while 32KD fragment drops notably. 17KD fragment quantity enhances remarkably in 180mmHg and 240mmHg group when compared with control and 120mmHg group. Conclusion:
1) As pressure elevating, MPV↓and PCDW↑; when cultured under 180mmHg hydrostatic pressure, as time progressing similar alterations shows up.
2) Higher hydrostatic pressures can boost the activated complex of GPIIb/GPIIIa and platelet aggregation.
3) High hydrostatic pressures can elevate P-slectin expression level; give rise to CD40L (CD154) splitted.
4) Under high hydrostatic pressure PPARγquantity has no remarkable changes but its activity increases to some extent as pressure elevates and time progress.
5) Hydrostatic pressures can activate Caspase 3 and cause platelet apoptosis. Section two: The effects of high hydrostatic pressure on megakaryocyte
Object:
To investigate the effects of high hydrostatic pressure on the morphrage, structure of megakaryocyte 01(Meg-01) and the expressions of platelet functional related gene, and approach the influence of high hydrostatic pressure on megakaryocyte function.
Method:
After the model of megakaryocyte culturing under high pressure has been constructed, Meg-01 cells were cultured under 4 different high pressures (0, 120, 180, 240mmHg), then cells proliferation and apoptosis percentage were measured by MTT and TUNEL respectively, the gene expressions of GPIIb, GPIIIa, P-selectin and PPARγwere measured by RT-PCR.
Result:
1) Proliferation rate: compared with control, cells had significant proliferations in 120mmHg group, but in higher pressure groups, 180mmHg and 240mmHg, the proliferation of cells were remarkable fewer than that of control and 120mmHg groups and in 240mmHg group cell proliferation rate was notably smaller than that of 180 mmHg group.
2) Apoptosis index: there was no significant difference in apoptosis index between 120mmHg and the control, but the apoptosis percentage of cells increased significantly in 180mmHg and 240mmHg groups.
3) mRNA volumes of GPIIb/ GPIIIa: mRNA levels of GPIIb and GPIIIa in higher hydrostatic pressure groups (180mmHg and 240mmHg) increased obviously when compared with the control and 120mmHg groups. And GPIIIa mRNA level in 240mmHg group was significant higher than that of 180mmHg group.
4) Volumes of P-selectin mRNA: P-selectin mRNA level in 180 mmHg group elevated significantly while in 240mmHg group it increased but with no significance.
5) Volumes of PPARγmRNA: compared with control, PPARγmRNA expression was stimulated obviously when treated with 120mmHg hydrostatic pressure, but there was no significant difference among 180mmHg, 240mmHg and the control groups.
Conclusion:
1) Moderate hydrostatic pressure (120mmHg) can stimulate Meg-01 proliferation, but higher hydrostatic pressures (180mmHg and 240mmHg) play the opposite role, inhibit proliferation and facilitate apoptosis.
2) Higher hydrostatic pressures (180mmHg and 240mmHg) can stimulate and promote the expressions of GPIIb/ GPIIIa and P-slectin in Meg-01.
3) 120mmHg hydrostatic pressure facilitates PPARγexpression but under higher hydrostatic pressures PPARγexpression mainly remains the same as usual. Section three: The role of PPARγin the protection of platelet under high hydrostatic pressures
Object:
To study the role of PPARγin the protection of platelet cultured in high hydrostatic pressure.
Method:
Platelets were divided into six groups randomly, C (normal atmosphere ), C+wy( interfered with PPARγagonist under normal atmosphere), C+gw( interfered with PPARγinhibitor under normal atmosphere), H ( high hydrostatic pressure, 180mmHg), H+wy(interfered with PPARγagonist under high hydrostatic pressure), H+gw(interfered with PPARγinhibitor under high hydrostatic pressure). Platelet aggregation rate, the concentration of calcium and NO, the expressions of iNOS、CD40L(CD154)、NFκB and P-selectin protein in platelet, NO levels in culture medium were measured after being cultured for 18h.
Result:
1) Platelet aggregation rates: Aggregation rates among C, C+wy and C+gw groups have no statistical difference; 2min, 4min and Max aggregation rates in group H are notable larger than those of C group; All three aggregation rates of group H+wy are significant smaller than group H; those of group H+gw is relatively higher (with no significance) than those of group H.
2) PAC-1: PAC-1 expressions had no statistical difference among C, C+wy and C+gw groups; PAC-1 expression in group H is remarkable higher than that of group C; PAC-1 expression in group C+wy is obvious fewer than that of group H; PAC-1 volume of group H+gw is larger than that of group H but with no statistical significance.
3)CD40L(CD154) expression: CD40L(CD154) expressions in both platelet membrane and cytoplasm have no statistical difference among C, C+wy and C+gw groups; CD40L(CD154) expression in platelet membrane of group H is notably higher than that of group C while when it comes to CD40L(CD154) expression in cytoplasm the opposite is true; CD40L(CD154) expression in platelet membrane of group H+wy is obviously fewer than that of group H while when it comes to CD40L(CD154) expression in cytoplasm the opposite is also true; CD40L(CD154) expression in platelet membrane of group H+gw is slightly more than that of group H while when it comes to CD40L(CD154) expression in cytoplasm the opposite is also true ,however, both difference is insignificant;
4) Calcium concentration in platelet: calcium concentrations have no statistical difference among C, C+wy and C+gw groups but calcium concentration in group H is remarkably higher than that of control group; calcium concentration in group H+wy, which is obviously smaller than that of group H, is still notably higher than that of control group; calcium concentration in group H+gw is higher than that of control and H+wy group, but the latter comparison had no significant difference. 5) NO levels in platelet and platelet upper clear solution: NO levels in platelet and platelet upper clear solution have no statistical difference among C, C+wy and C+gw groups; NO levels in platelet and platelet upper clear solution of group H are significantly lower than that of control; NO levels in platelet and platelet upper clear solution of group H+wy are remarkably higher than that of high hydrostatic pressure groups but when compared with control that still have remarkable decline; NO levels of group H+gw are obviously lower than group control and H+wy but have no significant difference when compared with group H.
6) expressions of eNOS and iNOS in platelet: eNOS and iNOS expressions in platelet have no statistical difference among C, C+wy and C+gw groups; when group H is compared with control, iNOS expression increases notably and eNOS expression decreases remarkably while when group H+wy compared with group H the opposite is true; eNOS expression in group H+gw is much fewer(but with no significance)than that of group H while iNOS expression in group H+gw is significantly higher than that of group H.
7) Expression of P-selectin: P-selectin expression has no statistical difference among C, C+wy and C+GW groups and H, H+wy and H+gw groups. P-selectin expression in group H is remarkably higher than that of control.
8) Expression of NF-κB: NF-κB expression has no statistical difference among C, C+wy and C+gw groups and H, H+wy and H+gw groups. NF-κB expression in group H is notably greater than that of control.
Conclusion:
1) After cultured for 18hrs under 180mmHg hydrostatic pressure, PPARγagonist Wy14643 can greatly reduce not only platelet aggregation and activation but also the expressions of PAC-1 and CD40L (CD154) in platelet membrane. This can be achieved by either way of reducing calcium concentration and increasing eNOS expression in platelet or decreasing iNOS expression and enhancing NO volume in platelet and culture medium.
2) After cultured for 18hrs under 180mmHg hydrostatic pressure, PPARγinhibitor Gw9662 can not further activate platelet which maybe because PPARγhas already been in the highly prohibitive state.
已经明确高血压病患者血小板聚集性和粘附性均明显亢进。由于相关研究多集中于临床观察,但高血压病患者常合并多种异常情况,除血压外,其他并存的因素也参与血小板激活。因此血压本身对血小板激活的作用及其程度尚不明确。为此,我们在离体孵育的条件下,观察静水压对血小板的影响。
最近研究发现,血小板不仅能合成蛋白质,还通过剪切预存于多聚核糖体中的未成熟的RNA为成熟的mRNA,翻译mRNA合成蛋白质。当血小板激活时起动了细胞内的信号转导反应,导致特异性的RNA被翻译并产生蛋白质。由于血小板源于巨核细胞,巨核细胞的mRNA也能直接影响血小板激活后的功能。我们设定,高血压病患者的血小板的异常变化,部分可能是巨核细胞变化的继发变化。目前,高血压对外周循环巨核细胞是否影响,是否会进而影响血小板的状态及功能,尚未见研究报道。
血小板含有高水平的过氧化物酶体增殖物激活受体γ(Peroxisome Proliferator Activated Receptor, PPARγ)。PPAR的作用广泛,涉及多方面。研究证实血小板的PPARγ具有活性,能抑制血小板的激活。对于高血压病患者血小板PPARγ的变化,尤其是PPARγ的信号转导途径,值得深入研究。
基于上述未决问题,本课题利用自制的高静水压装置,在体外利用不同的压力条件孵育血小板及巨核细胞,进行以下几方面的研究:高静水压对血小板的形态和结构及功能的影响、高静水压对巨核细胞的形态和结构及功能的影响和PPARγ在高静水压刺激血小板中信号传导作用。通过上述研究,以加深高血压对血小板激活认识,为临床更有效地防治高血压靶器官损害提供有价值的实验依据。
第一部份高静水压对血小板的形态和结构及功能的影响
目的:
利用体外静水压孵育血小板的模型,观察不同压力,以及同一压力不同时间对血小板的形态、结构及功能的影响。
方法:
建立高压培养的模型后,以180mmHg处理血小板不同时段(0h、1h、6h和18h)和不同静水压(0mmHg、120mmHg、180mmHg和240mmHg)处理血小板18小时,观察以下指标。1、在电镜下观察血小板超微结构的变化,全自动血细胞分析仪测定血小板形态参数,全自动生化分析仪测定血小板孵育液中LDH;2、测定血小板聚集率和流式细胞仪测定血小板活化表面标志物PAC-1和CD40L(CD154);3、免疫印迹法测定血小板内CD40L(CD154)、P-selectin、PPARγ和Caspase 3的蛋白表达;4、血小板PPARγ活性的测定。
结果:
1、血小板超微结构:正常大气压孵育血小板18小时,血小板细胞呈静止状态,细胞规整、表面突触少、致密颗粒多;高静水压刺激后,细胞形态大小不一,血小板羽状突起增多。细胞内致密颗粒减少,血小板呈活化状态,个别细胞出现消解、崩溃的图形。
2、血小板的参数:正常大气压孵育血小板不同时段的血小板的参数无明显差异。180mmHg静水压处理下6小时和18小时,平均血小板体积(MPV)显著减少,18小时血小板分布宽度(PDW)的显著增加。不同的静水压处理血小板,240mmHg静水压组血小板数(PLT)的显著降低;与对照相比,180mmHg和240mmHg引起血小板MPV、PDW的显著减少。
3、LDH的变化:正常大气压孵育各时段的上清中的LDH无统计学差异,而180mmHg处理组,随时间延长,LDH浓度逐渐增加,18小时段明显高于其他时段。不同静水压处理组,240mmHg、180mmHg处理组均明显高于对照组。
4、血小板聚集率:180mmHg刺激1小时后最大聚集率即明显增加,6小时和18小时的2min、4min和最大聚集率均有极显著的增加。不同压力组的比较可见,180mmHg和240mmHg处理组的2min、4min和最大聚集率均比对照组显著增加,180mmHg组的2min、4min和最大聚集率均显著高于120mmHg组,但240mmHg组的聚集率较180mmHg组有所下降,但无统计学意义。
5、血小板PAC-1:三个高静水压处理组血小板的PAC-1均显著增加;在180mmHg处理1小时血小板激活有增加但不显著,在6小时和18小时相较0小时和1小时均可检测到显著的血小板激活。
6、P-selectin蛋白表达:不同静水压的比较表明,随着静水压的增加,P-selectin表达逐渐增加。180mmHg和240mmHg的P-selectin的表达显著高于120mmHg组。
7、CD154的表达:血小板胞浆裂解液中CD40L(CD154)的量在180mmHg和240mmHg有显著性降低;流式细胞仪检测血小板膜表面CD40L(CD154)的表达随着静水压的增加而明显增加。
8、血小板内PPARγ的蛋白量:不同压力组之间,同一压力不同时间组之间的血小板内PPARγ的蛋白量无统计学差异。但PPARγ活性随着压力的增加(120mmHg和180mmHg)而显著增加,但240mmHg组PPARγ活性与180mmHg则明显的降低。
9、血小板内caspase 3的蛋白表达:随着压力的增加,血小板内caspase 3的17KD片段蛋白表达逐渐出现并增高,同时32KD的量显著性减少。180mmHg和240mmHg17KD的量比对照和120mmHg有显著增加。
结论:
1、随着压力增高,血小板形态发生明显改变,表现为平均血小板体积(MPV)显著减少,分布宽度的增加;在180mmHg静水压下,随着时间的延长也出现类似改变。
2、较高的高静水压(180mmHg和240mmHg)使活化的GPIIb/GPIIIa复合体(PAC-1)增多,促使血小板活化和聚集。
3、高静水压(180mmHg和240mmHg)可使血小板内P-slectin的表达量增加,其胞浆的CD40L(CD154)裂解,胞膜上CD40L(CD154)增加,血小板中炎症相关分子释放增加,促进炎症反应。
4、静水压刺激的血小板中PPARγ蛋白含量无明显变化,但其活性随着压力和时间的增加而有一定幅度的增加。
5、180mmHg和240mmHg刺激血小板18小时导致血小板破坏,机制与高静水压激活血小板Caspase 3,导致血小板凋亡有关。
第二部份高静水压对巨核细胞的影响
目的:
研究静水压对巨核细胞系Meg-01形态、结构及血小板功能相关基因表达的影响,探讨高静水压对巨核细胞功能的影响。
方法:
建立巨核细胞高压培养的模型,分别用不同的压力(0、120、180、240mmHg)刺激Meg-01细胞,用MTT法检测细胞的增殖率,TUNEL法检测细胞凋亡百分比,RTPCR测定GPIIb、GPIIIa、P-selectin和PPARγ基因表达。
结果:
1、增殖率:120mmHg组与正常对照组相比,细胞有明显增殖,180mmHg组和240mmHg组与对照相、120mmHg组比,细胞增殖率有明显下降,且180mmHg组和240mmHg组二组间的细胞增殖率也有显著性降低。
2、凋亡率:20mmHg处理组的凋亡指数与正常对照组无显著性差异,而静水压达180mmHg和240mmHg时细胞的凋亡率显著增加。
3、GPIIb/GPIIIa的mRNA的量高静水压处理巨核细胞系Meg-01细胞后,细胞内GPIIb的mRNA的量180mmHg和240mmHg处理组与正常对照组和120mmHg有明显增加。细胞内GPIIIa的mRNA的量180mmHg和240mmHg处理组与正常对照组和120mmHg有明显增加,240mmHg较180mmHg也有显著增加。
4、P-electin的mRNA量:P-electin的mRNA量在静水压180mmHg显著性增加,240mmHg虽有增加,但无显著性意义。
5、PPARγ的mRNA量:细胞在120mmHg刺激下,PPARγ的mRNA量有显著性增加,而在180mmHg和240mmHg的压力下PPARγ的mRNA量相较对照组无显著性变化。
结论:
1、120mmHg静水压可促进Meg-01细胞增殖,而180mmHg和240mmHg则抑制巨核细胞增殖,促进其凋亡。
2、180mmHg和240mmHg静水压刺激促进Meg-01细胞表达GPIIb/GPIIIa和P-slectin。
3、120mmHg静水压下PPARγ表达增强,在更高静水压则无变化。
第三部份PPARγ对高静水压处理血小板的保护作用
目的:
探讨PPARγ在高静水压处理的血小板中保护作用。
方法:
实验分为C、C+wy、C+gw、H、H+wy和H+gw六组(C为正常大气压孵育,H为180mmHg静水压孵育,wy为PPARγ激动剂,gw为PPARγ阻断剂),处理18小时后检测各组血小板聚集率,血小板内钙离子浓度,血小板内和孵育基中NO的量,血小板内血小板内的iNOS、eNOS、CD40L(CD154)、NFκB和P-selectin蛋白量的表达。
结果:
1、血小板聚集率:C、C+wy、C+gw三组的血小板聚集率无统计差别。H组2min、4min和Max聚集率均显著高于C组; H+wy组的三种聚集率显著低于H组;H+gw组比H组有升高,但无统计意义。2、PAC-1:C、C+wy、C+gw三组的血小板膜PAC-1无统计差别。H组的PAC-1比C组显著升高;H+wy组的PAC-1比H组显著降低;H+gw组的PAC-1较H组高,但无统计意义。
3、CD40L(CD154)的表达:C、C+wy、C+gw三组的血小板膜CD40L(CD154)和细胞内CD40L(CD154)的表达无统计差别。H组血小板膜上CD40L(CD154)的表达比C组显著升高,细胞内的CD40L(CD154)比C组明显降低;H+wy组血小板膜上CD40L(CD154)比H组显著降低,细胞内的CD40L(CD154)比C组明显升高;H+gw组血小板膜上的CD40L(CD154比H组高,细胞内的CD40L(CD154)比H组低,但均无统计意义。
4、血小板内钙离子浓度:C、C+wy、C+gw三组的血小板内钙离子浓度无统计差别。H组血小板内钙明显高于对照组;H+wy组的钙离子明显低于H组,但仍显著高于对照组;H+gw组的钙离子明显高于对照组,比H+wy组高,但无统计意义。
5、血小板的上清中和血小板内的NO:C、C+wy、C+gw三组的血小板的上清中和血小板内的NO无统计差别。H组血小板的上清中和血小板内的NO显著低于对照组;H+wy组和高静水压组相比NO均有显著性升高,和正常对照组相比上清和细胞内的NO仍有明显降低;H+gw组明显低于对照组和H+wy组,和H组无显著性差别。
6、血小板内iNOS和eNOS的表达:C、C+wy、C+gw三组的血小板内iNOS和eNOS的表达无统计差别。H组的iNOS明显高对照组,eNOS显著下降;H+wy组与H组相比,eNOS明显升高,iNOS显著下降;H+gw组eNOS较H组低,但无统计意义,iNOS明显高于H组。
7、血小板P-selectin、表达:C、C+wy、C+gw三组的血小板P-selectin的表达无统计差别。H组的P-selectin明显高于C组;H+wy组、H+gw组与H组相比, P-selectin表达无显著性差异。
8、血小板内NF-κB的表达:C、C+wy、C+gw三组的血小板NF-κB的表达无统计差别。H组的NF-κB明显高于以C组;H+wy组、H+gw组与H组相比, P-selectin表达无显著性差异。
结论:
1、在180mmHg刺激18小时的血小板,PPARγ的激动剂Wy 14643能显著降低血小板聚集和活化,降低血小板膜上PAC-1的表达,可能通过降低血小板内钙离子浓度,升高血小板内的eNOS的表达,降低iNOS的表达,而升高血小板内和孵育液中的NO的量这两条途径。
2、在180mmHg刺激18小时的血小板,PPARγ的阻断剂Gw 9662并不能进一步激活血小板,可能血小板中PPARγ已经处于高度抑制状态有关。
3、PPARγ的激动剂Wy 14643显著降低血小板膜上CD40L(CD154)的表达,稳定胞浆中的CD40L(CD154);而对P-selectin没有明显的改变,提示血小板内的PPARγ调节血小板的免疫功能可能主要通过调节CD40L的量来调节,而对P-slectin和NF-κB作用不显著。
Background and design
Findings have already revealed that both platelet clumping and cohesion are sthenic (overactive) in patients with hypertension. Pertinent researches on the relationship of hypertension and platelet mainly focus on clinical observation. However, other factors besides blood pressure can also be involved in platelet activation as hypertension patients often suffer from many other abnormal conditions. Thus, the effect of hydrostatic pressure on platelet was tested in vitro in order to determine platelet activation and its extent influenced by the single factor of blood pressure.
Recent researches found that platelets can not only synthesize proteins but also translate mRNA into proteins via splicing pre-mature mRNA pre-stored in polyribosome into mature mRNA. When Platelet activated, signal transduction reaction starts up and results in subsequent specific RNA translating and protein synthesis. As platelet originates from megakaryocyte, megakaryocyte mRNA has a direct influence on the functions of activated platelet. The changes of platelet in hypertension patients could be a subsequent event of megakaryocytes alternation. However, it has not been studied yet, whether high pressure can influence peripheral vascular megakaryocytes and further affect platelet states and functions.
Peroxisome proliferator activated receptors (PPARs), which have a wide range of diverse functions, expresses highly in Platelet. Studies showed that platelet PPARγis active and can prohibit the activation of platelet. Platelet PPARγchanges in hypertension patients and the signal transduction ways via which PPARγcan exert its influence deserve further study.
Based on the unresolved problems mentioned above, the present research firstly cultured platelet and megakaryocyte in vitro under different hydrostatic pressure(a self-designed high-hydrostatic-pressure device is made to treat platelet under different hydrostatic pressure) and then studied in the following respects: high hydrostatic pressure’s influence on the shape, structure and function of both platelet and megakaryocyte; the protection of PPARγafter platelet has been intrigued by high hydrostatic pressure. This study aims to enhance the understanding of activation of platelet under high blood pressure so as to evaluate therapeutic effect of antihypertensive in new perspective. The experimental data could provide valuable basis for more effectively clinical prevention and treatment of damage in target hypertensive organ
Section one: influence of high hydrostatic pressure on platelet shape, structure and function
Objective:
By utilizing the model of culturing platelet under high hydrostatic pressure in vitro and treating platelet under 180mmHg hydrostatic pressure for 4 different period of time(0h,1h,6h and 18h) or treating platelet for 18h but under 4 different hydrostatic pressure(0mmHg,120mmHg,180mmHg and 240mmHg), changes of platelet shape, structure and function have been studied.
Method:
Firstly the model of platelet culturing under high hydrostatic pressure has been established. Then platelet has been treated under 180mmHg hydrostatic pressure for 4 different period of time(0h, 1h, 6h and 18h) or treated for 18h but under 4 different hydrostatic pressure (0mmHg, 120mmHg, 180mmHg and 240mmHg), and the following indices has been tested: 1) platelet ultrastructure changes were observed by electron microscope, platelet shape parameters were determined by full automatic blood cell analyzer, LDH in platelet culture medium detected by full automatic biochemical analyzer; 2) platelet aggregation rate, 3) PAC-1 and CD40L(CD154) (surface marker of platelet activation) determined by flow cytometry; 4) the expression of CD40L(CD154), P-selectin, PPARγand Caspase3 in platelet determined by immunoblot; 5) PPARγactivity.
Results:
1) Platelet ultrastructure: after 18h culturing under normal atmosphere pressure, platelet cells are of the similar shape and size and in a static state with a small number of dendritic extensions and many dense granule while after high hydrostatic pressure treatment, platelet cells become different in shape and size with an increasing number of dendritic extensions. With the decrease of dense granule platelet becomes active and some specific platelet cells take on a picture of breakdown.
2) Platelet parameters: have no significant difference among various time-treatment groups (all under the same atmosphere pressure). When treated for 6h or 18h under180mmHg hydrostatic pressure the mean platelet volume(MPV) declined dramatically , the platelet component distribution width(PDW) rose remarkably in the 18h time-treatment group; among the different hydrostatic pressure treatment groups, PLT dropped essentially in 240mmHg treatment group; compared with the control group, MPV and PDW decreased dramatically when treated in 180 mmHg and 240mmHg.
3) LDH changes: when platelet cultured under normal atmosphere pressure LDH in upper clear solution among different time-treatment groups have no statistic difference. However, when cultured under180mmHg hydrostatic pressure, LDH concentration increases gradually and its value in 18h time-treatment group is remarkably higher than other groups. Among different hydrostatic pressure treatment groups, LDH concentration in both 240mmHg and 180mmHg treatment groups is notably higher than control group.
4) Platelet aggregation rate: after 1 hour treatment of 180mmHg hydrostatic pressure the largest aggregation rate enhances greatly and after 6 hour or 18 hour treatment the 2min, 4min and largest aggregation rate elevate significantly. Among different hydrostatic pressure groups (180mmHg and 240mmHg), the 2min, 4min and largest aggregation rate increased significantly and all three aggregation rates of 180mmHg group are significant higher than those of 120mmHg group. However, the maximal aggregation rate of 240mmHg group is smaller than that of 180mmHg group but with no statistical significance.
5) Platelet PAC-1: PAC-1concentration in all three hydrostatic pressure groups elevate greatly; after 1 hour treatment of 180mmHg hydrostatic pressure, the activation of platelets increase but not significant while after 6 hour or 18 hour treatment remarkable platelet activation can be detected when compared with 0 hour or 1 hour treatment.
6) As the elevation of hydrostatic pressure, the volume of both P-selectin protein increases gradually: P-selectin expression level in 180mmHg and 240mmHg group increases more dramatically than that of 120mmHg group.
7) CD154 expression: The quantity of CD40L (CD154) in platelet lyses in 180mmHg and 240mmHg group declines notably; Tests by flow cytometry show that CD40L (CD154) expression in platelet surface increases remarkably with hydrostatic pressure elevating.
8) PPARγexpression in platelet: PPARγexpression levels among different pressure-treat groups and different time-treat groups show no statistical difference. Yet, PPARγactivity augmented remarkably when pressure rose from 120mmHg to 180 mmHg. However, PPARγactivity under 240mmHg is greatly lower than that of 180mmHg group.
9) Expression level of caspase 3 in platelet: with pressure elevation 17KD fragment of caspase 3 gradually emerges and accumulates while 32KD fragment drops notably. 17KD fragment quantity enhances remarkably in 180mmHg and 240mmHg group when compared with control and 120mmHg group. Conclusion:
1) As pressure elevating, MPV↓and PCDW↑; when cultured under 180mmHg hydrostatic pressure, as time progressing similar alterations shows up.
2) Higher hydrostatic pressures can boost the activated complex of GPIIb/GPIIIa and platelet aggregation.
3) High hydrostatic pressures can elevate P-slectin expression level; give rise to CD40L (CD154) splitted.
4) Under high hydrostatic pressure PPARγquantity has no remarkable changes but its activity increases to some extent as pressure elevates and time progress.
5) Hydrostatic pressures can activate Caspase 3 and cause platelet apoptosis. Section two: The effects of high hydrostatic pressure on megakaryocyte
Object:
To investigate the effects of high hydrostatic pressure on the morphrage, structure of megakaryocyte 01(Meg-01) and the expressions of platelet functional related gene, and approach the influence of high hydrostatic pressure on megakaryocyte function.
Method:
After the model of megakaryocyte culturing under high pressure has been constructed, Meg-01 cells were cultured under 4 different high pressures (0, 120, 180, 240mmHg), then cells proliferation and apoptosis percentage were measured by MTT and TUNEL respectively, the gene expressions of GPIIb, GPIIIa, P-selectin and PPARγwere measured by RT-PCR.
Result:
1) Proliferation rate: compared with control, cells had significant proliferations in 120mmHg group, but in higher pressure groups, 180mmHg and 240mmHg, the proliferation of cells were remarkable fewer than that of control and 120mmHg groups and in 240mmHg group cell proliferation rate was notably smaller than that of 180 mmHg group.
2) Apoptosis index: there was no significant difference in apoptosis index between 120mmHg and the control, but the apoptosis percentage of cells increased significantly in 180mmHg and 240mmHg groups.
3) mRNA volumes of GPIIb/ GPIIIa: mRNA levels of GPIIb and GPIIIa in higher hydrostatic pressure groups (180mmHg and 240mmHg) increased obviously when compared with the control and 120mmHg groups. And GPIIIa mRNA level in 240mmHg group was significant higher than that of 180mmHg group.
4) Volumes of P-selectin mRNA: P-selectin mRNA level in 180 mmHg group elevated significantly while in 240mmHg group it increased but with no significance.
5) Volumes of PPARγmRNA: compared with control, PPARγmRNA expression was stimulated obviously when treated with 120mmHg hydrostatic pressure, but there was no significant difference among 180mmHg, 240mmHg and the control groups.
Conclusion:
1) Moderate hydrostatic pressure (120mmHg) can stimulate Meg-01 proliferation, but higher hydrostatic pressures (180mmHg and 240mmHg) play the opposite role, inhibit proliferation and facilitate apoptosis.
2) Higher hydrostatic pressures (180mmHg and 240mmHg) can stimulate and promote the expressions of GPIIb/ GPIIIa and P-slectin in Meg-01.
3) 120mmHg hydrostatic pressure facilitates PPARγexpression but under higher hydrostatic pressures PPARγexpression mainly remains the same as usual. Section three: The role of PPARγin the protection of platelet under high hydrostatic pressures
Object:
To study the role of PPARγin the protection of platelet cultured in high hydrostatic pressure.
Method:
Platelets were divided into six groups randomly, C (normal atmosphere ), C+wy( interfered with PPARγagonist under normal atmosphere), C+gw( interfered with PPARγinhibitor under normal atmosphere), H ( high hydrostatic pressure, 180mmHg), H+wy(interfered with PPARγagonist under high hydrostatic pressure), H+gw(interfered with PPARγinhibitor under high hydrostatic pressure). Platelet aggregation rate, the concentration of calcium and NO, the expressions of iNOS、CD40L(CD154)、NFκB and P-selectin protein in platelet, NO levels in culture medium were measured after being cultured for 18h.
Result:
1) Platelet aggregation rates: Aggregation rates among C, C+wy and C+gw groups have no statistical difference; 2min, 4min and Max aggregation rates in group H are notable larger than those of C group; All three aggregation rates of group H+wy are significant smaller than group H; those of group H+gw is relatively higher (with no significance) than those of group H.
2) PAC-1: PAC-1 expressions had no statistical difference among C, C+wy and C+gw groups; PAC-1 expression in group H is remarkable higher than that of group C; PAC-1 expression in group C+wy is obvious fewer than that of group H; PAC-1 volume of group H+gw is larger than that of group H but with no statistical significance.
3)CD40L(CD154) expression: CD40L(CD154) expressions in both platelet membrane and cytoplasm have no statistical difference among C, C+wy and C+gw groups; CD40L(CD154) expression in platelet membrane of group H is notably higher than that of group C while when it comes to CD40L(CD154) expression in cytoplasm the opposite is true; CD40L(CD154) expression in platelet membrane of group H+wy is obviously fewer than that of group H while when it comes to CD40L(CD154) expression in cytoplasm the opposite is also true; CD40L(CD154) expression in platelet membrane of group H+gw is slightly more than that of group H while when it comes to CD40L(CD154) expression in cytoplasm the opposite is also true ,however, both difference is insignificant;
4) Calcium concentration in platelet: calcium concentrations have no statistical difference among C, C+wy and C+gw groups but calcium concentration in group H is remarkably higher than that of control group; calcium concentration in group H+wy, which is obviously smaller than that of group H, is still notably higher than that of control group; calcium concentration in group H+gw is higher than that of control and H+wy group, but the latter comparison had no significant difference. 5) NO levels in platelet and platelet upper clear solution: NO levels in platelet and platelet upper clear solution have no statistical difference among C, C+wy and C+gw groups; NO levels in platelet and platelet upper clear solution of group H are significantly lower than that of control; NO levels in platelet and platelet upper clear solution of group H+wy are remarkably higher than that of high hydrostatic pressure groups but when compared with control that still have remarkable decline; NO levels of group H+gw are obviously lower than group control and H+wy but have no significant difference when compared with group H.
6) expressions of eNOS and iNOS in platelet: eNOS and iNOS expressions in platelet have no statistical difference among C, C+wy and C+gw groups; when group H is compared with control, iNOS expression increases notably and eNOS expression decreases remarkably while when group H+wy compared with group H the opposite is true; eNOS expression in group H+gw is much fewer(but with no significance)than that of group H while iNOS expression in group H+gw is significantly higher than that of group H.
7) Expression of P-selectin: P-selectin expression has no statistical difference among C, C+wy and C+GW groups and H, H+wy and H+gw groups. P-selectin expression in group H is remarkably higher than that of control.
8) Expression of NF-κB: NF-κB expression has no statistical difference among C, C+wy and C+gw groups and H, H+wy and H+gw groups. NF-κB expression in group H is notably greater than that of control.
Conclusion:
1) After cultured for 18hrs under 180mmHg hydrostatic pressure, PPARγagonist Wy14643 can greatly reduce not only platelet aggregation and activation but also the expressions of PAC-1 and CD40L (CD154) in platelet membrane. This can be achieved by either way of reducing calcium concentration and increasing eNOS expression in platelet or decreasing iNOS expression and enhancing NO volume in platelet and culture medium.
2) After cultured for 18hrs under 180mmHg hydrostatic pressure, PPARγinhibitor Gw9662 can not further activate platelet which maybe because PPARγhas already been in the highly prohibitive state.
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